Figure 5.
Figure 5. PKC expression, contractile function, and phosphorylation in young and aging F344BN rats after gene transfer. (A) Representative Western blot detecting PKCα in NT myocytes and after AdGFP gene transfer along with PKCαDN after AdPKCαDN gene transfer in myocytes from 6- and 26-mo-old rats. A portion of silver-stained gel also is included as a loading control. (B) Representative Western blots showing gene transfer of AdPKCα or AdPKCαDN increases PKCα or PKCαDN expression, respectively, compared with NT myocytes. These representative blots also illustrate that gene transfer produces similar increases in PKCαDN expression in 6-mo-old (left panel) versus 26-mo-old (right panel) rat myocytes. As shown here and in E, the levels of PKCα expression after gene transfer of AdPKCα also were similar in 6- and >26-mo-old myocytes. These Western blots also were reprobed for PKCδ and ε expression in 6- and ≥26-mo-old myocytes. (C) Quantitative analysis of PKCαDN/PKCα protein expression after gene transfer. In the left panel, PKCαDN protein expression after gene transfer is normalized to a silver-stained gel band from young (6 mo) and older (≥26 mo) samples. Results are expressed as mean + SEM in C–E, and n = number of rat hearts in C and E. PKCαDN expression is similar in myocytes from the two age groups when compared using an unpaired Student’s t test (P > 0.05). Gene transfer also produced similar increases in PKCα expression in myocytes from 6- and ≥26-mo-old rats (data not shown). Thus, expression in the two age groups was pooled to quantitatively compare the level of PKCα versus PKCαDN expression levels after gene transfer and relative to NT myocytes using one-way ANOVA and post hoc Dunnett’s tests (*, P < 0.05 versus NT; right panel). This comparison shows gene transfer results in comparable increases in PKCα and PKCαDN expression in adult myocytes. (D) Sarcomere length, peak shortening amplitude, and the rates of shortening and relengthening in NT myocytes and after gene transfer of AdPKCα or AdPKCαDN for 6–18- versus ≥26-mo-old rats (n = number of myocytes). A two-way ANOVA (PKC, age) and post hoc Tukey’s tests were used for the statistical comparison (*, P < 0.05 versus NT for PKC effect; ♦, P < 0.05 versus 6–18-mo-old myocytes for age effect). (E) Representative Western analysis of PKCα, p-PLB, and PLB expression plus a silver-stained portion of the gel in 6- and 26-mo-old rat myocytes after AdPKCα or AdPKCαDN gene transfer compared with NT myocytes (left, middle panel). Quantitative analysis of p-PLB/PLB ratio in 6–18- versus ≥26-mo-old rat myocytes (right panel) after PKCαDN gene transfer is compared using a Student’s unpaired t test (P > 0.05; right panel). (F) Representative Western blots show cTnI p-S23/24, p-S45, and total cTnI expression in 6-, 26-, and 34-mo-old rat myocytes after AdGFP or AdPKCαDN gene transfer compared with time-matched NT myocytes.

PKC expression, contractile function, and phosphorylation in young and aging F344BN rats after gene transfer. (A) Representative Western blot detecting PKCα in NT myocytes and after AdGFP gene transfer along with PKCαDN after AdPKCαDN gene transfer in myocytes from 6- and 26-mo-old rats. A portion of silver-stained gel also is included as a loading control. (B) Representative Western blots showing gene transfer of AdPKCα or AdPKCαDN increases PKCα or PKCαDN expression, respectively, compared with NT myocytes. These representative blots also illustrate that gene transfer produces similar increases in PKCαDN expression in 6-mo-old (left panel) versus 26-mo-old (right panel) rat myocytes. As shown here and in E, the levels of PKCα expression after gene transfer of AdPKCα also were similar in 6- and >26-mo-old myocytes. These Western blots also were reprobed for PKCδ and ε expression in 6- and ≥26-mo-old myocytes. (C) Quantitative analysis of PKCαDN/PKCα protein expression after gene transfer. In the left panel, PKCαDN protein expression after gene transfer is normalized to a silver-stained gel band from young (6 mo) and older (≥26 mo) samples. Results are expressed as mean + SEM in C–E, and n = number of rat hearts in C and E. PKCαDN expression is similar in myocytes from the two age groups when compared using an unpaired Student’s t test (P > 0.05). Gene transfer also produced similar increases in PKCα expression in myocytes from 6- and ≥26-mo-old rats (data not shown). Thus, expression in the two age groups was pooled to quantitatively compare the level of PKCα versus PKCαDN expression levels after gene transfer and relative to NT myocytes using one-way ANOVA and post hoc Dunnett’s tests (*, P < 0.05 versus NT; right panel). This comparison shows gene transfer results in comparable increases in PKCα and PKCαDN expression in adult myocytes. (D) Sarcomere length, peak shortening amplitude, and the rates of shortening and relengthening in NT myocytes and after gene transfer of AdPKCα or AdPKCαDN for 6–18- versus ≥26-mo-old rats (n = number of myocytes). A two-way ANOVA (PKC, age) and post hoc Tukey’s tests were used for the statistical comparison (*, P < 0.05 versus NT for PKC effect; ♦, P < 0.05 versus 6–18-mo-old myocytes for age effect). (E) Representative Western analysis of PKCα, p-PLB, and PLB expression plus a silver-stained portion of the gel in 6- and 26-mo-old rat myocytes after AdPKCα or AdPKCαDN gene transfer compared with NT myocytes (left, middle panel). Quantitative analysis of p-PLB/PLB ratio in 6–18- versus ≥26-mo-old rat myocytes (right panel) after PKCαDN gene transfer is compared using a Student’s unpaired t test (P > 0.05; right panel). (F) Representative Western blots show cTnI p-S23/24, p-S45, and total cTnI expression in 6-, 26-, and 34-mo-old rat myocytes after AdGFP or AdPKCαDN gene transfer compared with time-matched NT myocytes.

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